Controlling motivation for cocaine is the goal of long-term treatment success of cocaine addiction, which may require reversal of drug-induced changes in gene expression. Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in addictive behaviors has received little attention. RNA binding proteins and microRNAs serve as master switches controlling gene expression, with mRNA stability estimated to control about 20% of brain-expressed genes. Our research suggests that the RNA binding protein HuD and the microRNA miR-495 play opposite roles in the control of addiction-related gene expression and behavior: 1) They are predicted to bind the same GU-rich sequence in mRNAs; 2) Their binding sites are overrepresented in transcripts from an addiction-related gene (ARG) database; 3) They show differential regulation by cocaine in addiction-related brain regions, with miR-495 being downregulated and HuD upregulated; 3) In vitro manipulations of these molecules result in opposite effects on the expression of two of their target genes, BDNF and arc; 5) Most importantly, in vivo manipulations of these molecules show contrasting effects on motivation for cocaine. Based upon these results, we hypothesize that HuD and miR- 495 play a role in drug abuse by post-transcriptionally competing for binding to the same sequences and controlling the expression of ARGs in opposing directions. To test this hypothesis, we will: 1) test the functional competition between HuD and miR-495 for a) specific mRNA binding sites and the control of ARG gene expression, and b) cocaine conditioned place preference in mice overexpressing HuD in forebrain neurons; 2) determine the effects of viral-mediated gene transfer of miR- 495 and 3) HuD to the nucleus accumbens shell of rats using the following 3 models of motivation for cocaine: i) break point on a progressive ratio schedule of cocaine reinforcement, ii) extinction of cocaine-seeking behavior, and iii) reinstatement of extinguished cocaine-seeking behavior; 4) examine changes in the levels of miR-495 and HuD and selected target genes, including BDNF and arc in rats which have been manipulated to express varying degrees of motivation for cocaine. The proposed work synergistically combines the expertise of Dr. Perrone-Bizzozero in mRNA stability, HuD function, and target analyses and Dr. Neisewander in animal models of addiction and the neurocircuitry involved. The outcome of this work will provide new knowledge about the post-transcriptional mechanisms regulating addiction-related gene expression, an exciting new area of neuroscience research. A better understanding of these regulatory mechanisms is a pre-requisite for the application of these new tools in addiction research and ultimately in the treatment of this disorder. PUBLIC HEALTH RELEVANCE: Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in the establishment of addictive behaviors has received very little attention. Therefore, characterizing the competing roles of mR-495 and HuD in the control of addiction-related genes will unveil a new mechanism underlying the maladaptive changes in synaptic plasticity during drug addiction and provide potential new targets for intervention.